Platelet-derived growth factor analogues

ABSTRACT

This invention relates to cyclic platelet-derived growth factor (PDGF) analogues and pharmaceutical compositions thereof.

This application is a divisional of Ser. No. 08/635,007 filed Apr. 19,1996, which is a 371 of PCT/GB 94/02331 filed Oct. 21, 1994.

FIELD OF THE INVENTION

This invention relates to platelet-derived growth actor (PDGF) analoguesand their use as cell antiproliferative agents.

BACKGROUND OF THE PRIOR ART

Relevant background material is incorporated herein by reference in thetext to the listed references in the appended bibliography.

Platelet-derived growth factor (PDGF) is a potent mitogen for connectivetissue cells and promotes the proliferation of fibroblasts and smoothmuscle cells (SMC) [33]. The growth factor is a 28-31 KD dimeric, highlybasic (Pi=9.8-10) glycoprotein consisting of two highly homologous (upto 60% sequence homology) polypeptide chains which are the products ofdistinct genes. The gene products designated A (on chromosome 7) and B(on chromosome 22) are assembled to form either a disulphide-linkedheterodimer (PDGF-AB) or a homodimer (PDGF-AA or PDGF-BB). Analysis ofthe PDGF present in human platelets reveals that it is a mixture of allthree dimeric forms with AB being the predominant form (up to 70%)[10;12]. The human prot-oncogene, c-sis, which codes for the PDGF-Bchain [21] has been identified as the human homologue of the v-sisoncogene of the transforming retrovirus, simian sarcoma virus. Thisoncogene codes for the protein p28 v-sis which has been identified asPDGF-BB [5].

The cloning and amino acid sequencing of the A and B chains of humanPDGF have shown that both chains are synthesised as precursor moleculeswith hydrophobic leader sequences and both chains undergo proteolyticcleavage at the N-termini during maturation. The B chain is alsoprocessed at the C-terminal end [21:20].

The three isoforms of PDGF exert their biological effects by bindingwith different affinities to two different receptor types, denoted α andβ. Ligand binding induces dimerization of receptors; the A-subunit ofPDGF binds to α-receptors whereas the B-subunit binds to both α- andβ-receptors [2].

When PDGF dimer is treated with reducing agents, the protein loses itsbiological activity irreversibly, suggesting that the proteinconformation is disturbed by reduction of critical disulphide bonds[16]. PDGF has 8 cysteine residues which are highly conserved betweenthe two chains. Six residues are involved in 3 intra-moleculardisulphide bonds: Cys-16 - - - Cys-60, Cys-49 - - - Cys-97 andCys-53 - - - Cys-99. The other two cysteine residues are involved inasymmetrical inter-molecular disulphide bonds, Cys-43 - - - Cys-52 [11].

A systematic analysis of the abilities of different peptides, derivedfrom the PDGF-B chain sequence, to compete with ¹²⁵ I-PDGF-BB forbinding to PDGF β-receptors, has led to the identification of tworegions in the B-chain corresponding to amino acid residues 35-40 and78-83 that seem important for receptor binding. A peptide correspondingto the two sequences (ANFLVW - - - EIVRKKP) (SEQ ID NOS: 12 and 13,respectively) has been found to be effective as an antagonist for PDGF,although detailed analysis has shown the pure peptide to be less active[6].

Site-directed mutagenesis studies, using deletion and substitutionmutants of PDGF-BB or of the homologous v-sis gene as well as PDGF-A/Bchimeras, have also identified a number of amino acid residues which areimportant for the biological activity of PDGF. The region Ile-25 - - -Phe-38 has been identified as a binding domain by site directedmutagenesis of the v-sis gene [9]. Amino acid residue Asn-34 has beenfound to be essential for the PDGF-B-like transforming efficiency ofPDGF-A/B chimera [27]. Using a different functional assay, which selectsfor mutants with reduced binding to both receptor types, Ile-30 and, toa lesser extent, Arg-27 have been shown to be important [3]. Basicpolypeptides such as polylysine and protamine sulphate inhibit PDGFbinding to its receptor, suggesting a role for ligand positive charge inthe binding interaction. A receptor binding domain has been assigned toa region at the C-terminal end which is rich with basic amino acid,residues Lys-80 - - - Cys-97 [39]. This region contains the sequenceVal-78 - - - Arg-79 - - - Lys-80 - - - Lys-81 - - - Pro-82 (SEQ ID NO:14), which is conserved in both the A and B chains, and therefore may beinvolved in the binding of both chains to PDGF α-receptor. A mutantPDGF-A chain in which the cationic sequence Arg-Lys-Lys has beenreplaced by the sequence Glu-Glu-Glu displays a marked reduction in bothbinding affinity for PDGF α-receptor and mitogenic activity infibroblast cells [7]. Initial studies with neutralizing monoclonalantibodies raised to PDGF-BB indicates that the segment between Thr-20and Cys-43 represents a surface domain of PDGF-BB and contains aminoacid residues involved in receptor binding [22].

Recently, the crystal structure of the homodimeric BB isoform of humanrecombinant PDGF has been determined [26]. The protein polypeptide chainis folded into two highly twisted anti-parallel pairs of β-strands andcontains an unusual knotted arrangement of three intramoleculardisulphide bonds. Dimerization leads to the clustering of three surfaceloops at each end of the elongated dimer, which most probably form thereceptor recognition sites. The three loops are: loop I: Ile-25 - - -Leu-38, loop II: Cys-53 - - - Val-58 and loop III: Val-78 - - - Lys-81.

Antibodies to PDGF would be extremely useful in the study of PDGFprocessing and biosynthesis. It has been difficult to make high avidityantibodies against PDGF, maybe because the molecule is conserved betweenspecies and only recently have monoclonal antibodies against PDGF becomeavailable [22;34;12;38]. Rabbit and goat antisera to PDGF have been madeto the two chains using protein purified from human platelets orrecombinant protein or synthetic peptides, some showing chainspecificity and neutralizing activity [28;17;13;37;30]. None of theantibodies raised to peptides however have been capable of recognisingthe native molecule or able to neutralize its biological activities.

PDGF has been implicated in many biological systems. Originally, theclose similarity between PDGF and the transforming factor involved inSSV transformation led to the concept that over-production of the factorwas involved in the development of human malignancies [14]. Examinationof many tumour cell lines shows that the A and B chains are commonlyexpressed in such cell lines [15;24]. In general, aberrant expression ofPDGF or of PDGF receptors is likely to be involved in the stimulation ofthe growth of certain tumours. In addition, over-activity of PDGF couldalso be part of the development of certain non-malignant disordersinvolving an excess of cell proliferation. Examples includeatherosclerosis, where PDGF-induced stimulation of smooth muscle cellproliferation could contribute to the thickening of the intima ofaffected vessels [32], as well as chronic fibrotic processes, where PDGFcould be involved in the stimulation of connective tissue cellproliferation. Ferns et al [8] showed that in a rat experimental modelof angioplasty, polyclonal antibodies to PDGF administered intravenouslyinhibited smooth muscle cell accumulation in the intima of injuredarteries, while administration of PDGF induced SMC proliferation in themedia by 2-3 fold and, more significantly, increased SMC migration fromthe media to the intima by 20-fold [19].

However, PDGF does have a normal function. PDGF and PDGF receptors areexpressed in embryonic tissues and in the placenta [23;18] whichsuggests a function for PDGF during development. A role for. PDGF inneuronal development has also been proven [25] and PDGF and itsreceptors are present in the peripheral and central nervous systems[40;36]. PDGF is known to stimulate growth as well as chemotaxis ofconnective tissue cells and also chemotaxis of inflammatory cells, whichsuggests a role in wound healing [4;35]. Recently, PDGF has been used ina clinical trial to look at its wound healing capability. Locallyapplied PDGF stimulates the healing of large bed sores [31]. PDGFβ-receptors occur on capillary endothelial cells [29] and PDGF has weakangiogenic activity [29] which may suggest that its stimulatory effectis important in wound healing.

The varied roles of PDGF, both beneficial and adverse, make PDGFagonists and antagonists highly desirable. They can be used as areplacement for PDGF in wound healing or as inhibitors of the adverseeffects of PDGF. Antibodies with neutralizing activity, whether to themitogenic effect of PDGF and/or the chemotactic effect can also beuseful as inhibitors of PDGF adverse effects.

Accordingly, in one aspect the present invention provides novel PDGFpeptide analogues and compositions consisting of or containing them foruse as antiproliferative agents, particularly antiatherosclerotic,antiatherogenetic, anti-inflammatory or antifibrotic agents. Theinvention also provides such novel PDGF peptide analogues andcompositions consisting of or containing them for use as PDGF agonistsfor use in wound healing.

Particular PDGF analogues according to the present invention areidentified in Table 1 hereinbelow (SEQ ID NOS: 1-7). Preferably, thePDGF peptide analogues of the invention, as prepared and used in otheraspects and embodiments of the invention, are greater than about 90%pure, more preferably greater than about 95% pure, even more preferablygreater than about 99% pure.

Pharmaceutical compositions in accordance with the present inventionpreferably comprise one or more of the PDGF analogues of the inventiontogether with a pharmaceutically acceptable diluent and/or carrier.Suitable carriers/diluents are well known in the art and include salineor other sterile aqueous media, optionally including additionalcomponents such as buffer salts and preservatives, or sugars, starches,salts or mixtures thereof.

Peptides according to the present invention may be provided for use inany suitable form appropriate to the protocol of administration and/orthe needs of a patient.

Apart from the pharmaceutically acceptable compositions referred toabove, the peptides may for example be provided, either singly or incombination, in lyophilized or freeze dried solid forms.

Within the scope of the invention are linked peptides comprising a firstanalogue selected from the group consisting of GP1 (SEQ ID NO: 1), GP2(SEQ ID NO: 1), GP3 (SEQ ID NO: 2), GP4 (SEQ ID NO: 2), GP9 (SEQ ID NO:3) and GP10 (SEQ ID NO: 3) (as identified in Table 1 hereinbelow) and asecond peptide analogue selected from the group consisting of GP5 (SEQID NO: 4), GP6 (SEQ ID NO: 4), GP7 (SEQ ID NO: 4), GP8 (SEQ ID NO: 4),GP21a (SEQ ID NO: 4), GP21 (SEQ ID NO: 4) and GP22 (SEQ ID NO: 7) (asidentified in Table 1 hereinbelow),

The invention further provides the novel PDGF peptide analogues for usein assays and kits for assays.

It is to be understood that within the scope of the present inventionare peptide analogues as described and identified herein in which one ormore amino acids are substituted with other amino acids, or in whichthere is inserted a spacer, for example a dithiol group or a diaminogroup or multiples of amino acid residues, e.g. glycine, as shown inTable 2 hereinbelow, peptides GP11 (SEQ ID NO: 8), GP12 (SEQ ID NO: 8),GP13 (SEQ ID NO: 9) and GP14 (SEQ ID NO: 9). The spacer may also be ahomo- or hetero-bifunctional crosslinker, for example theheterobifunctional crosslinkerN-(4-carboxy-cyclohexyl-methyl)-maleimide, as shown in Table 3hereinbelow, peptides GP20 and GP23 (SEQ ID NOS: 10 and 11,respectively), providing generally of course that the essential activityof the peptide remains substantially unchanged.

The invention further provides the synthesis and use of cyclic peptidessuch as those derived from GP4 (SEQ ID NO: 2) and GP8 (SEQ ID NO: 5) asshown in Table 4 below, peptides GP24 (SEQ ID NO: 1) and GP25 (SEQ IDNO: 6).

The invention further provides the novel PDGF peptide analogues for usein assays and kits for assays, either in the free form or linked to acarrier molecule such as a protein or a solid particle, as well asmodified peptides with e.g. biotin or fluorescein isothiocyanate, suchas those shown in Table 5 hereinbelow, peptides GP15 (SEQ ID NO: 1),GP16 (SEQ ID NO: 2), GP19 (SEQ ID NO: 3), GP17 (SEQ ID NO: 4) and GP18(SEQ ID NO: 5).

In a second aspect, the present invention provides a method ofinhibiting or stimulating cell proliferation, particularly smooth musclecell, 3T3-fibroblast cell, connective tissue cell or inflammatory cellproliferation, by use or administration, particularly to a host, of aneffective amount of a PDGF peptide analogue as defined above.

The invention further provides a method of inhibiting or stimulatingPDGF-induced DNA synthesis comprising use or administration, such as toa host, of an effective amount of a PDGF peptide analogue as definedabove.

In a further aspect, the present invention provides PDGF peptideanalogues as defined above for use in inhibiting or stimulating growthand/or chemotaxis of cells such as those identified above.

In yet a further aspect, the present invention provides theabove-defined PDGF peptide analogues, particularly the linked peptideanalogues of the invention, for use as immunogens for the production ofpolyclonal and monoclonal antibodies to PDGF, especially for diagnostic,prognostic and therapeutic uses. Such methods of production ofpolyclonal and monoclonal antibodies are also within the scope of theinvention.

In yet another aspect of the present invention, the novel PDGF analoguesare provided for and used in methods of inhibiting PDGF-induced DNAsynthesis, for example by use of or administration of an effectiveamount of one or more of the above defined PDGF peptide analogues.

Administration of peptides of the invention in any of the methodsdescribed herein may be via any suitable protocol. Preferably,administration to a host, especially a human host, is by intravenousinjection or infusion, and may be systemic or topical. Suchadministration of peptides of the invention is in such an amount as togive the desired effective result of the peptide's activity at theintended site. Thus, a quantity which constitutes an "effective" amountmay depend upon various parameters, such as body weight of the patient,degree of activity required, intended site of activity, severity of thecondition to be treated or prevented, all of which will be wellunderstood and appreciated by persons skilled in the art.

Generally, an amount (or total amount) of peptide will be administeredwhich gives a concentration in plasma of from about 1 to about 100 mgml⁻¹, more preferably from about 1 to about 10 mg ml⁻¹.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in further detail, withreference to the accompanying drawings, in which:

FIG. 1 shows relative mitogenic effects of various PDGF relatedpeptides;

FIGS. 2A and 2B show the results of a ¹²⁵ I-PDGF binding assay, asdescribed further below;

FIGS. 3A and 3B show the results of titrations of, respectively,anti-Tg-GP4 vs.GP4 and anti-Tg-GP8 vs.GP8;

FIGS. 4A and 4B show the results of titrations of, respectively,anti-Tg-GP4 vs.PDGF-BB and anti-Tg-GP4 vs.FGF and EGF;

FIGS. 5A and 5B show the results of titrations of selected poly- andmonoclonal antibodies by direct ELISA against PDGF-BB;

FIG. 6 shows the inhibition of radiolabelled PDGF-BB binding to humansmooth muscle cells by anti-peptide antibodies; and

FIGS. 7A and 7B, 8A and 8B, and 9A and 9B show the HPLC and massspectroscopy profiles of peptides GP4, GP8 and GP14, respectively (SEQID NOS: 2, 5 and 9 respectively).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Methods

1) Synthesis of PDGF-BB Peptide Analogues

A series of PDGF-BB related peptides were synthesised, with or withoutmodifications, by solid-phase on a Milligen 9050 Pepsynthesizer, usingthe FMOC-polyamide continuous method, as listed in Table 1 hereinbelow.

Acetylation of the N-terminal end of the peptides was performed afterthe completion of the synthesis. The resin was acetylated on thesolid-support with 45% acetic anhydride in dimethylformamide.Deprotection and cleavage of the resin were carried out in the normalmanner.

Biotinylation and FITC labelling were carried out while the peptideswere still attached to the resin and prior to deprotection.Biotin-caproate-N-hydroxysucccinimide (B-NHS) and fluoresceinisothiocaynate were used to label the free N-terminal end of thepeptides.

All peptides were purified to at least 95% homogeneity by HPLC and theirmolecular weights determined by mass spectroscopy. FIGS. 7, 8 and 9 showexamples of the HPLC and mass spectroscopy profiles of peptides GP4, GP8and GP14, respectively (SEQ ID NOS: 2, 5 and 9 respectively).

2) Effect of PDGF Peptides on Fibroblast Cells in Culture

The stimulatory or inhibitory effect of the peptides on the murinefibroblast cell line Swiss 3T3.A31 were investigated using the [³H]-thymidine uptake assay as described by Raines & Ross [28].

3) Effect of PDGF Peptides on ¹²⁵ I-PDGF-BB Binding to 3T3 Cells andHuman Smooth Muscle Cells

PDGF-BB binding inhibition assay was performed as described by Engstromet al [6]. A murine fibroblast cell line 3T3.A31 and human aortic smoothmuscle cells were used.

4) Production of Antisera to PDGF-Peptides

Rabbits and mice were immunised with the peptides either in the freeform mixed with Freund's adjuvant or conjugated to a carrier protein(Thyroglobulin or keyhole haemocyanin). Antisera were tested forantibody production to the peptides and PDGF using ELISA, dot blotassays and SDS-PAGE followed by Western blotting.

5) Effect of Anti-PDGF peptides antibodies on ¹²⁵ I-PDGF binding toHuman Smooth Muscle cells

The IgGs of the polyclonal anti-PDGF peptides antisera were purifiedfrom the antisera by affinity chromatography on a protein G-Sepharosecolumn as described by the manufacturers (Pharmacia, Uppsala, Sweden).The effect of the IgG on the binding of radiolabelled PDGF-BB to humansmooth muscle cells was investigated using essentially the sameprocedure as for the peptides (method 3 above). In the test, peptideswere replaced with IgG.

Results

The peptides were tested for their ability to stimulate thymidine uptakein the cells in culture.

FIG. 1 shows an example of the results obtained with some of thepeptides. Peptide GP4 (SEQ ID NO: 2) showed the highest stimulatoryeffect acting as an agonist for PDGF-BB. The mitogenic effect of GP4 wasalmost completely abolished upon reduction and alkylation of theC-terminal end cysteine residue. This strongly suggests that the peptideis acting via the formation of a dimeric form during the incubation withthe cells and that it is the dimerisation which produces the increase inthe stimulatory activity. This conclusion is also supported by the lowstimulatory effect of peptide GP2 (SEQ ID NO: 1) which has the sameamino acid sequence as GP4 but without the C-terminal cysteine.

Peptide GP8 (SEQ ID NO: 5) was not as stimulatory as GP4 (SEQ ID NO: 2).

Some of the peptides were tested for their ability to inhibit thebinding of radiolabelled PDGF-BB to 3T3 cells. Both GP4 (SEQ ID NO: 2)and GP8 (SEQ ID NO: 5) showed modest inhibition of binding at theconcentrations tested, as illustrated in FIG. 2A. Peptides GP20 and GP14(SEQ ID NOS: 10 and 9, respectively) were potent inhibitors of labelledPDGF binding to human smooth muscle cells, as shown in FIG. 2B.

Rabbits immunised with GP4 and GP8 peptides (SEQ ID NOS: 2 and 5,respectively) linked to thyroglobulin produced high titre antibodies tothe corresponding immunising peptide as determine by ELISA, asillustrated in FIGS. 3A and 3B.

One of the rabbits immunised with GP4 (SEQ ID NO: 2) also producedantibodies reactive with native PDGF-BB, and had no cross reactivitywith human recombinant fibroblast growth factor (FGF) and epidermalgrowth factor (EGF). This is illustrated in FIG. 4.

Tables 6, 7 and 8 hereinbelow summarise the results of immunochemicalcharacterisation of polyclonal and monoclonal antisera raised toPDGF-derived peptides.

Western immunoblot analysis of polyclonal antisera reactivity withnative and reduced PDGF-BB (Table 6) shows that peptides GP4 and GP21a(SEQ ID NOS: 2 and 6, respectively) produced antibodies that reactedwith the native PDGF. The competitive ELISA data are shown in Table 7.15 monoclonal antibody hybridomas raised to peptide GP4 (SEQ ID NO: 2)coupled to thyroglobulin were immunochemically characterised as shown inTable 8. FIGS. 5A and 5B show typical titration curves for polyclonaland monoclonal antisera against PDGF-BB.

The IgG fraction from rabbits immunised with peptides GP4 and GP21a (SEQID NOS: 2 and 6, respectively) were effective in inhibiting the bindingof radio-labelled PDGF-BB to human smooth muscle cells in culture, asshown in FIG. 6.

                  TABLE 1                                                         ______________________________________                                        PDGF-B CHAIN PEPTIDES                                                         ______________________________________                                        LOOP I                                                                        .sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38                                                               GP1                                                Ac-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38                                                            GP2                                                .sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38 -C                                                            GP3                                                Ac-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38 -C                                                         GP4                                                .sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L-V-W-P-P-C.sup.43                                                     GP9                                                Ac-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L-V-W-P-P-C.sup.43                                                  GP10                                               LOOP III                                                                      .sup.73 R-K-I-E-I-V-R-K-K.sup.81                                                                         GP5                                                Ac-.sup.73 R-K-I-E-I-V-R-K-K.sup.81                                                                      GP6                                                .sup.73 R-K-I-E-I-V-R-K-K.sup.81 -C                                                                      GP7                                                Ac-.sup.73 R-K-I-E-I-V-R-K-K.sup.81 -C                                                                   GP8                                                .sup.73 R-K-I-E-I-V-R-K-K-P-I-F-K-K-A-T-V.sup.89                                                         GP21a                                              .sup.73 R-K-I-E-I-V-R-K-K-P-I-F-K-K-A-T-V.sup.89 -C                                                      GP21                                               Ac-.sup.73 R-K-I-E-I-V-R-K-K-P-I-F-K-K-A-T-V.sup.89 -C                                                   GP22                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        PDGF-B CHAIN PEPTIDES                                                         (LOOP I & LOOP III using Glvcvl spacers)                                      ______________________________________                                        .sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38 -(G-G-G-G)-                                                     GP11                                             .sup.73 R-K-I-E-I-V-R-K-K.sup.81 -C                                           Ac-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38 -(G-G-G-G)-                                                  GP12                                             .sup.73 R-K-I-E-I-R-K-K.sup.81 -C                                             .sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38 -(G-G-G-G-G-G)-                                                 GP13                                             .sup.73 R-K-I-E-I-V-R-K-K.sup.81 -C                                           Ac-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38 -(G-G-G-G-G-G)-                                              GP14                                             .sup.73 R-K-I-E-I-V-R-K-K.sup.81 -C                                           ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        CROSS-LINKED PDGF LOOP I & LOOP III PEPTIDES                                  ______________________________________                                        Ac-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L-V-W-P-P-C.sup.43 -(SMCC)-                                           GP20                                             .sup.73 R-K-I-E-I-V-R-K-K.sup.81 -C                                           Ac-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38 -C-(SMCC)-                                                   GP23                                             .sup.73 R-K-I-E-I-V-R-K-K.sup.81 -C                                           ______________________________________                                         {SMCC:N-(4-carboxy-cyclohexyl-methyl)-maleimide OR any heterobifunctional     crosslinker                                                              

                  TABLE 4                                                         ______________________________________                                        CYCLIC PDGF-B CHAIN PEPTIDES                                                  ______________________________________                                        LOOP I                                                                         ##STR1##                                                                     GP24                                                                          LOOP III                                                                       ##STR2##                                                                     GP25                                                                          ______________________________________                                         (Xa = bridging spacer arm)                                                    aa1 = amino acid/acids of Cterminus                                           aa2 = amino acid/acids of Nterminus                                      

                  TABLE 5                                                         ______________________________________                                        AFFINITY-LABELLED PDGF-B CHAIN PEPTIDES                                       ______________________________________                                        LOOP I                                                                        X-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38                                                              GP15                                              X-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L.sup.38 -C                                                           GP16                                              X-.sup.25 I-S-R-R-L-I-D-R-T-N-A-N-F-L-V-W-P-P-C.sup.43                                                    GP19                                              LOOP III                                                                      X-.sup.73 R-K-I-E-I-V-R-K-K.sup.81                                                                        GP17                                              X-.sup.73 R-K-I-E-I-V-R-K-K.sup.81 -C                                                                     GP18                                              ______________________________________                                         (X = Biotin or FITC)                                                     

                                      TABLE 6                                     __________________________________________________________________________    Polyclonal anti-PDGF peptides antisera analysis by western Blot                          vs PDGF                                                                            vs PDGF                                                                             vs PDGF                                                                             vs RED-PDGF                                                                          vs RED-PDGF                                Antibody                                                                           Immunogen                                                                           Ser-1/100                                                                          Ser-1/1000                                                                          Ser-1/10000                                                                         Ser-1/100                                                                            Ser-1/10000                                __________________________________________________________________________    Rb 86                                                                              GP4   -    -     -     ++     -                                          Rb 65                                                                              Tg-GP4                                                                              ++++ +++   -     +++++  +++++                                      Rb 66                                                                              Tg-GP4                                                                              +++  +++   -     +++++  +++                                        Rb 109                                                                             GP10  -    -     -     ++     -                                          Rb 37                                                                              GP10  -    -     -     +++    ++                                         Rb 38                                                                              Tg-GP10                                                                             -    -     -     ++++   +++                                        Rb 39                                                                              Tg-GP10                                                                             -    -     -     +++++  ++++                                       Rb 112                                                                             Tg-GP10                                                                             -    -     -     +++    +                                          Rb 67                                                                              Tg-GP8                                                                              -    -     -     ++++   ++                                         Rb 68                                                                              Tg-GP8                                                                              +    -     -     ++     -                                          Rb 78                                                                              GP21a +++++                                                                              +++++ +     +++++  +++++                                      Rb 91                                                                              GP21a -    -     -     +++ (1/200)                                                                          + (1/20,000)                               Rb 113                                                                             Tg-GP4                                                                              - (1/200)                                                                          -     -     - (1/200)                                                                            - (1/20,000)                               Rb 114                                                                             Tg-GP4                                                                              ++ (1/200)                                                                         -     -     ++++ (1/200)                                                                         + (1/20,000)                               __________________________________________________________________________     +++++ Very strong                                                             ++++ Strong                                                                   +++ Medium                                                                    ++ Weak                                                                       + Very weak                                                                   - Negative                                                               

                                      TABLE 7                                     __________________________________________________________________________    Competitive ELISA analysis of polyclonal anti-PDGF-BB peptides antisera       Antibody                                                                           Immunogen                                                                           titre                                                                              GP4 IC50                                                                           GP10 IC50                                                                           GP21a IC50                                                                          GP8 IC50                                                                           PDGF IC50                               __________________________________________________________________________    Rb 86                                                                              GP4                                                                      Rb 65                                                                              Tg-GP4                                                                              1/243,000                                                                           3 nM                                                                               3 nM NONE  >6000 nM                                                                           180 nM                                  Rb 66                                                                              Tg-GP4                                                                              1/27,000                                                                            <2 nM                                                                             <2 nM NONE  NONE NONE                                    Rb 109                                                                             GP10  1/10,000                                                                            2 nM                                                                               4 nM NONE  NONE NONE                                    Rb 37                                                                              GP10  1/27,000                                                                            20 nM                                                                             10 nM NONE  NONE NONE                                    Rb 38                                                                              Tg-GP10                                                                             1/27,000                                                                           150 nM                                                                             74 nM NONE  NONE NONE                                    Rb 39                                                                              Tg-GP10                                                                             1/100,000                                                                           2 nM                                                                               2 nM NONE  NONE NONE                                    Rb 112                                                                             Tg-GP10                                                                             1/243,000                                                                           2 nM                                                                               3 nM NONE  NONE NONE                                    Rb 67                                                                              Tg-GP8                                                                              1/243,000                                                                          Not Sig                                                                            Not Sig                                                                             Not Sig                                                                                 2 nM                                                                           200 nM                                  Rb 68                                                                              Tg-GP8                                                                              1/243,000                                                                          Not Sig                                                                            Not Sig                                                                             Not Sig                                                                               <2 nM                                                                             30 nM                                  Rb 78                                                                              GP21a 1/15,000                                                                           NONE NONE  100 nM                                                                              NONE NONE                                    Rb 91                                                                              GP21a ND   ND   ND    ND    ND   ND                                      Rb 113                                                                             Tg-GP4                                                                              1/100,000                                                          Rb 114                                                                             Tg-GP4                                                                              1/2,000                                                            __________________________________________________________________________     Peptides tried up to 6000 nM, PDGF up to 200 nM                          

                                      TABLE 8                                     __________________________________________________________________________    Reactivities of monoclonal antibodies to peptide GP4 sub-class, ELISA,        CELIA and Western blot analysis                                                          ELISA                                                                             ELISA                                                                             BLOT  BLOT                                                                              CELIA                                                                             CELIA                                                                             CELIA                                                                             CELIA                                                                             CELIA                            ANTIBODY                                                                            Sub-class                                                                          TITRE                                                                             PDGF *                                                                            RED PDGF                                                                            PDGF                                                                              GP4 GP10                                                                              GP21a                                                                             GP8 PDGF **                          __________________________________________________________________________     1 DMB                                                                              IgG1 ND                                                                 ve                 +     -   -   -   -   -   -                                 2 DMB                                                                              IgG1 ND                                                                 ve    +    -    2 uM                                                                              2 uM -   -   -                                             3 DMB                                                                              IgG1 ND                                                                 ve    +    -   -   -     -   -   -                                             4 DMB                                                                              IgG1 ND                                                                 ve    +++  -   150 nM                                                                            150 nM                                                                              -   -   -                                             9 DB-1                                                                             IgG1 1/10                                                               ve    -    -    1 uM                                                                              1.2 uM                                                                             -   -    +10%?                                       10 DB-1                                                                             IgG1 1/243                                                                             10% +++++ -   400 nM                                                                             >6 uM                                                                            -   -    +10%?                           11 DB-1                                                                             IgM  1/2 30% ++    -   -   -   -   -   +175%?                           12 DB-1                                                                             IgG1 1/243                                                              ve    +++++                                                                              -    2 uM                                                                             -     -   -   -                                            13 DB-1                                                                             IgG1 1/10                                                               ve    -    -   200 nM                                                                            400 nM                                                                              -   -   -                                            15 DB-1                                                                             IgM  1/9 31% ++    -   -   -   -   -   +356%?                           17 DB-1                                                                             IgG1 1/81                                                                              30% +++++ -   180 nM                                                                             3 uM                                                                             -   -    +25%?                           19 DB-1                                                                             IgG1 1/1000                                                             ve    -    -    18 nM                                                                             18 nM                                                                              -   -    +10%?                                       21 DB-1                                                                             IgG1 1/1000                                                             ve    -    -    18 nM                                                                             30 nM                                                                              -   -   -                                            22 DB-1                                                                             IgG1 1/1000                                                             ve    -    -    20 nM                                                                             25 nM                                                                              -   -   -                                            __________________________________________________________________________     * Expressed as a percentage of OD given by 500 ng/ml Rb antiPDGF (Bachem)     ** An increase in signal may be caused by crosslinking                        In CELIAS, peptides tried up to 6000 nM, PDGF up to 200 nM               

REFERENCES

1. Bar, R. S. et al (1989) Endocrinology, 124, 1841-1848.

2. Claesson-Welsh, L. (1993) Cytokines, 5, 31-43.

3. Clements, X. et al (1991) EMBO J., 10, 4113-4120.

4. Deuel, T. F., Senior, R. M., Huang, J. S. & Griffin, G. L. (1981) J.Clin. Invest., 69, 1046-1049.

5. Doolittle, R. F., Hunkapiller, M. W., Hood, L. E. & 4 others (1983)Science, 221, 275-277.

6. Engstrom, U., Engstrom, A., Ernlund, A., Westermark, B. & Heldin, C-H. (1992) J. Biol. Chem., 267, 16581-16587.

7. Fenstermaker, R. A. et al (1993) J. Biol. Chem., 268, 10482-10489.

8. Ferns, G. A. A. et al (1991) Science, 253, 1129-1132.

9. Giese, N. A., LaRochelle, W. J., May-Siroff, M., Robbins, K. C. &Aaronson, S. A. (1990) Mol. Cell Biol., 10, 5496-5501.

10. Hammacher, A., Hellman, U., Johnsson, A., Osttman, A., Gunnarsson,K., Westermark, B., Wasteson, A. & Heldin,C- H. (1988) J. Biol. Chem.,263, 16493-16498.

11. Haniu,M., Rohde, M. F. & Kenney, W. C. (1993) Biochemistry, 32,2431-2437.

12. Hart, C. E., Bailey, M., Curtis, D. A., Osborn, S., Raines, E.,Ross, R. & Forstorm, J. W. (1990) Biochemistry, 29, 166-172.

13. Huang, J. S., Huang, S. S. &Deuel, T. F. (1983) J. Cell Biol., 97,383-388.

14. Heldin, C- H. (1992) EMBO J., 11, 4251-4259.

15. Heldin, C- H. & Westermark, B. (1989) British Med. Bull., 45,453-464.

16. Heldin, C- H., Westermark, A., & Wasteson, A. (1981) Exp. Cell Res.,136, 255-261.

17. Heldin, C- H., Westermark, A. & Wasteson, A. (1981) Proc. Natl.Acad. Sci., 78, 3664-3668.

18. Holmgren, L., Claesson-Welsh, L., Heldin, C- H. & Ohlsson, R. (1992)Growth Factors, 6, 219-232.

19. Jawein, A. et al (1992) J. Clin. Invest., 89, 507-511.

20. Johnsson, A., Betsholtz, C., Heldin, C. H. & Westermark, B. (1986)EMBO J., 5, 1535-1541.

21. Joseph, S. F., Guo, C., Ratner, L. & Wong-Staal, F. (1984) Science,223, 487-490.

22. LaRochelle, W., Robbins, K. C. & Aaranson, S. A. (1989) Mol. Cell.Biol., 9, 3538-3542.

23. Mercola, M. et al (1990) Dev. Biol., 138, 114-122.

24. Nister, M. et al (1988) Cancer Res., 48, 3910-3918.

25. Noble, M. et al (1988) Nature, 333, 560-562.

26. Oefner, C. et al (1992) EMBO J., 11, 3921-3926.

27. Ostman, A., Andersson, M., Hellman, U. & Heldin, C- H. (1991) J.Biol. Chem., 266, 10073-10077.

28. Raines, E. W. & Ross, R. (1982) J. Biol. Chem., 257, 5154-5160.

29. Risau, W. (1992) Growth Factors, In Press.

30. Robins, K. C. et al (1983) Nature, 305, 605-609.

31. Robson, M. C. et al (1992) Lancet, 339, 23-25.

32. Ross, R. (1993) Nature, 362, 801-809.

33. Ross, R., Raines, E. W. & Bowen-Pope, D. F. (1986) Cell, 46,155-169.

34. Shiraishi, T. et al (1989) Clin. Chim. Acta, 184, 65-74.

35. Siegbhan, A., Hammacher, A., Westermark, B. & Heldin, C- H. (1990) JClin. Invest., 85, 916-920.

36. Smits, A. et al (1991) Proc. Natl. Acad. Sci., 88, 8159-8163.

37. Thyberg, J. et al (1990) J. Cell Sci., 97, 219-229.

38. Vassbotn, F. S., Langeland, N., Hagen, I. & Holmsen, A. (1990)Biochem. Biophys. Acta, 1054, 246-249.

39. Vogel, S. & Hoppe, J. (1989) Biochemistry, 28, 2961-2966.

40. Yeh,H. J. et al (1991) Cell, 64, 209-216.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 14                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                  -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Ile may be acetylated":                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1                                                     #/note= "A cyclic peptide may be                                                             formed by - # linking Ile 1 with Leu 14 via a bridging                        spacer ar - #m"                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Binding-site                                                    (B) LOCATION: 1                                                     #/note= "Ile may be bound to biotin                                                          or FITC"                                                       #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Al - #a Asn Phe Leu                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Ile may be acetylated":                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Binding-site                                                    (B) LOCATION: 1                                                     #/note= "Ile may be bound to biotin                                                          or FITC"                                                       #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Al - #a Asn Phe Leu Cys             #                15                                                           - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Ile may be acetylated":                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Binding-site                                                    (B) LOCATION: 1                                                     #/note= "Ile may be bound to biotin                                                          or FITC"                                                       #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Al - #a Asn Phe Leu Val Trp         #                15                                                           - Pro Pro Cys                                                                 - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Arg may be acetylated":                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Binding-site                                                    (B) LOCATION: 1                                                     #/note= "Arg may be bound to biotin                                                          or FITC"                                                       #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Arg Lys Ile Glu Ile Val Arg Lys Lys                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Arg may be acetylated":                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Binding-site                                                    (B) LOCATION: 1                                                     #/note= "Arg may be bound to biotin                                                          or FITC"                                                       #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Arg Lys Ile Glu Ile Val Arg Lys Lys Cys                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                  -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1                                                     #/note= "A cyclic peptide may be                                                             formed by - # linking Arg 1 with Val 17 via a bridging                        spacer ar - #m"                                                #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Arg Lys Ile Glu Ile Val Arg Lys Lys Pro Il - #e Phe Lys Lys Ala Thr         #                15                                                           - Val                                                                         - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 18 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Arg may be acetylated":                                              #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Arg Lys Ile Glu Ile Val Arg Lys Lys Pro Il - #e Phe Lys Lys Ala Thr         #                15                                                           - Val Cys                                                                     - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 28 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Ile may be acetylated":                                              #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Al - #a Asn Phe Leu Gly Gly         #                15                                                           - Gly Gly Arg Lys Ile Glu Ile Val Arg Lys Ly - #s Cys                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 30 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Ile may be acetylated":                                              #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Al - #a Asn Phe Leu Gly Gly         #                15                                                           - Gly Gly Gly Gly Arg Lys Ile Glu Ile Val Ar - #g Lys Lys Cys                 #            30                                                               - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 29 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 19..20                                                #/note= "Cross-linker may beION:                                                             N-(4-carboxy - #-cyclohexyl-methyl)-maleimide or any                          other het - #erobifunctional cross-linker."                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Ile may be acetylated":                                              #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Al - #a Asn Phe Leu Val Trp         #                15                                                           - Pro Pro Cys Arg Lys Ile Glu Ile Val Arg Ly - #s Lys Cys                     #            25                                                               - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 25 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 15..16                                                #/note= "Cross-linker may beION:                                                             N-(4-carboxy - #-cyclohexyl-methyl)-maleimide or any                          other het - #erobifunctional cross-linker."                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Ile may be acetylated":                                              #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ile Ser Arg Arg Leu Ile Asp Arg Thr Asn Al - #a Asn Phe Leu Cys Arg         #                15                                                           - Lys Ile Glu Ile Val Arg Lys Lys Cys                                         #            25                                                               - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ala Asn Phe Leu Val Trp                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Glu Ile Val Arg Lys Lys Pro                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Val Arg Lys Lys Pro                                                         1               5                                                             __________________________________________________________________________

We claim:
 1. A platelet-derived growth factor peptide analogueconsisting of an amino acid sequence selected from the group consistingof: ##STR3## wherein the peptide analogue is in cyclized form.
 2. Aplatelet-derived growth factor peptide analogue consisting of an aminoacid sequence:Wherein: Xa=a bridging spacer arm; aa1=amino acid/acids ofthe C-terminus; and aa2=amino acid/acids of the N-terminus.
 3. Thepeptide analogue of claim 1, which has a purity greater than 90%.
 4. Thepeptide analogue of claim 2, which has a purity greater than 90%.
 5. Thepeptide analogue of claim 1, which has a purity greater than 95%.
 6. Thepeptide analogue of claim 2, which has a purity greater than 95%.
 7. Apharmaceutical composition comprising one or more peptide analoguesaccording to claim 1 together with a pharmaceutically acceptable diluentand/or carrier.
 8. A pharmaceutical composition according to claim 7,wherein the peptide analogue(s) is present in an amount such as to givea concentration thereof in plasma of a host to which the composition isadministered of from 1 to 100 mg/ml.